THE EXTRACTION, PURIFICATION AND EVALUATION

OF COMPOUNDS FROM THE LEAVES OF LEONOTIS

LEONORUS FOR ANTICONVULSANT ACTIVITY

Theoneste MUHIZI

A thesis submitted in partial fulfilment of requirements for the degree of

Master of Science in the Department of Chemistry, University of the

Western Cape

Supervisors: -Professor Ivan R. Green

Department of Chemistry

-Professor George J. Amabeoku

Department of Pharmacology

May, 2002

11

In memory of my late dear parents Emmanuel N. and Catherine N.

To my dear wife, Charlotte U. and my daughters,

Annick Mireille M.M.andAnge Joseline M.G. for their patience

III

THE EXTRACTION, PURIFICATION AND EVALUATION OF COMPOUNDS

FROM THE LEAVES OF LEONOTIS LEONORUS FOR ANTICONVULSANT

ACTIVITY

KEYWORDS

Medicinal plants

Leonotis leonorus

Natural products

Epilepsy

Antiepileptic components

Pharmacological test

Extraction

Purification

Chromatography

Evaluation

Test tube reactions

Soectroscooic methods

IV

ABSTRACT

THE EXTRACTION, PURIFICATION AND EVALUATION OF COMPOUNDS

FROM THE LEAVES OF LEONOTIS LEONORUS FOR ANTICONVULSANT

ACTIVITY

Theoneste MUHIZI

MSc thesis, Department of Chemistry, University of the Western Cape,

The aim of this study is to isolate and evaluate the anticonvulsant components

from the leaves of Leonotis leonorus (L) R.aR. and to see if there is any

change in activity with the origin of the plant material and I or the season in

which plant material is collected. Therefore, in this study, two sites were

chosen for collection of plant material and the collection was made in summer

and in winter.

Chemical, physical and pharmacological methods were used to isolate,

identify and to evaluate compounds isolated from the leaves of Leonotis

leonorus for anticonvulsant activity.

Tonic seizures were chemically induced in mice using pentylenetetrazole

(PTZ: 95mg/kg, ip). Different extracts of the plant such as hexane, methanol,

and aqueous were tested for anticonvulsant activities. The ~rude aqueous

extract (100-400 mg/kg, ip) significantly delayed the onset of PTZ (95 mg/kg,

ip) induced tonic seizures with 400 mg/kg (ip) protecting 37.5% of the animals

against the seizures. Similarly crude methanol extracts (100-400 mg/kg, ip)

significantly delayed the onset of tonic seizures induced by PTZ (95 mg/kg, ip)

with 100 mg/kg (ip) of the crude methanol extract of plant collected from Cape

Flats Nature Reserve protecting 50 % of mice against the seizures. The crude

hexane extract and aqueous extract obtained from the residue after methanol

extraction did not significantly affect the onset of seizures elicited by PTZ (95

mg/kg, ip) or alter the incidence of the seizures in all doses used. All plant

material used in the above investigation was collected during the summer

months. Doses of 100-400 mg/kg (ip) of crude methanol extract of plants

collected during the winter months also significantly delayed the onset of PTZ

(95 mg/kg, ip) elicited seizures in mice but did not affect the incidence of the

seizures to any significant extent. Additionally, 100-400 mg/kg (ip) of isolated

fractions in the crude methanol extract significantly delayed the onset of PTZ

(95 mg/kg, ip)-induced seizures while 200-400 mg/kg (ip) of the fraction

significantly reduced the incidence of the seizures. On the other hand,

200 mg/kg (ip) and 400 mg/kg (ip) of the further purified component protected

75 % and 87.5 % of mice respectively against the seizures.

Spectra showing a characteristic profile of active components mixture from

methanol extract of Leonotis leonorus (L) R.aR. were obtained with IR,

GC/MS and NMR spectroscopy. Phytochemical analysis revealed that the

plant contains chemical constituents such as alkaloids, tannins, terpenoids

It is suspected that the terpenoid lactone and quinonoidand quinones.

components possess the antiepileptic propreties of Leonotis leonorus.

May, 2002

VI

DECLARATION

I declare that" The Extraction, Purification and Evaluation of compounds from

the leaves of Leonotis leonorus for anticonvulsant activity" is my own work,

that it has not been submitted for any degree or examination in any other

University, and that all the sources I have used or quoted have been indicated

and acknowledged by means of complete references.

Theoneste MUHIZI May, 2002

VII

ACKNOWLEDGEMENTS

wish to thank to the following persons who have actively contributed to the

achievement of this work:

Professor Ivan Robert Green and Professor George Jimboyeka Amabeoku

who accepted to be supervisors of this present work. Their remarks, advice,

guidance and dynamism enabled me to be well grounded in phytotherapy.

National University of Rwanda, which financially supported my work.

Especially Dr Emile Rwamasirabo, Rector of National University of Rwanda

and Dr Butera Jean Bosco, the Academic Vice Rector of National University

of Rwanda for giving me this opportunity to undertake this postgraduate study.

Mr Franz Weitz and Mrs Dawn Faroe, for the identification and donation of the

plant material respectively, used for my experiment.

Bienvenu E. for site identification of plant and his constant encouragement.

Mr Timmy Lesch, Mr Yusufu Alexander, Ms Celeste Farmer, Mr Brian Minnis

for their technical assistance, their human kindness shown to me throughout

the period of working on this project.

My colleagues of Chemistry and Pharmacology departments especially Ms

Natasha October, Ms Rene Pearce and Ms Lenah Lebelo for their helpful

remarks during my experiment,

My wife, Charlotte Uwampeta, for her constant and superb encouragement

and support, without which it could not have been possible to achieve this

goal. Her patience during my studies is very much appreciated.

VIII

CONTENTS

~

iiiKeywords

Abstract. . iv

Declaration vi

Acknowledgements... . vii

Contents, viii

List of tables. xii

xiiiList offigures List of abbreviations and symbols.

xiv

Chapter 1 INTRODUCTION. . 1

LITERATURE REVIEW.Chapter 2 5

2.1 General view on traditional medicine 6

2.1.1 Definitions 6

72.1.2 Advantages and disadvantages of traditional medicine

2.2 Natural products 10

2.2.1 General and .definitions 10

102.2.2 Classification of natural products.

15

18

19

2.2.3 Summary of natural products biogenesis """"""""'."

2.2.4 The accumulation factors of natural products in plants...

2.2.5 Utilization of natural products.. 2.2.6 Isolation of natural compounds.. "'"

21

2.2.7 Identification of natural compounds.. 24

IX

25

25

2.3 Synopsis of Leonotis leonorus (L) R.BR 2.3.1 Description and classification 2.3.2 Indications of Leonotis leonorus

26

272.3.3 Some compounds isolated from Leonotis ssp

2.4 General view on epilepsy 29

29

30

2.4.1 Definitions 2.4.2 Causes of convulsion

2.4.3 Impact of epilepsy in society 31

2.4.4 The pharmacological evaluation of anticonvulsants ... 31

Chapter 3 DESCRIPTION OF THE PROJECT 3.1 Introduction 33

34

353.2 Hypothesis and objectives of the project.

Chapter 4 METHODOLOGY 36

374.1 Preparation of plant material. 4.1.1 Collection

37

374.1.2 Drying of plant material.

4.1.3 Preparation of extracts 37

4.2 Pharmacological tests.. 40

4.2.1 Animals 4.2.2 Drugs and chemicals ". 40

40

4.2.3 Anticonvulsant activity assessment. 40

424.3 Isolation of active compounds

42

42

43

4.3.1 Choice of solvents """ 4.3.2 Detection of spots 4.3.3 Isolation of active compounds

x

4.4 Purification of components with anticonvulsant activity 46

48

48

50

4.5 Characterisation of active compounds... ...

4.5.1 Characterisation using coloured reactions.

4.5.2 Characterisation by spectroscopic method

4.6 Pharmacological results analysis. 50

Chapter 5 RESULTS 51

52

52

53

5.1 Extracts obtained from fine powder ..' 5.2 Yield obtained after extraction by fractionation 5.3 Characterisation of compounds obtained from methanol

extract (ME1,ME3) 5.4 Yields of anticonvulsant agents... 55

5.5 Chemical identification from test tube reactions 55

5.6 Convulsant activity of pentylenetetrazole... 56

5.7 Anticonvulsant activities of crude extracts 5.7.1 Effects of hexane, methanol, and aqueous extracts on 57

57PTZ- induced seizures 5.7.2 Effects of methanol extract (ME2) on PTZ-induced

60seizures. 5.8 Effects of different fractions of methanol extract on

PTZ- induced seizures... 61

63

5.9 Effects of two purified products (P1 and P2) obtained

from methanol extract on PTZ- induced seizures... ...

5.10 Effects of phenobarbitone and diazepam on

PTZ-induced seizures 64

Xl

5.11 Spectra obtained from two active compounds ... 65

Chapter 6 DISCUSSIONS AND CONCLUSIONS 71

6.1 Discussions 72

6.2 Conclusions 78

REFERENCES 79

XII

LIST OF TABLES

.E:99§

Table 1: Some examples of drugs from plant kingdom.. 9

Table 2: Characteristics of compounds found in methanol

extracts (ME1. ME3) 53

Table 3: Compounds detected in different fractions of methanol extract... 56

Table 4: Convulsant activity of pentylenetetrazole in mice 57

Table 5: Effects of hexane (HE), methanol (ME1. ME3) and aqueous

(AQ, AQM) extracts on PTZ- induced seizures in mice , 59

Table 6: Effects of methanol extract (ME2) on PTZ-induced seizures

in mice

Table 7: Effects of different fractions, F. F2, F3, F4 and Fs, on

PTZ-induced seizures in mice.. 62

Table 8: Effects of two purified products, P1 and P2, obtained from

methanol extract on PTZ induced-seizures in mice 63

Table 9: Effects of phenobarbitone and diazepam on PTZ- induced

64seizures in mice

XIII

LIST OF FIGURES

~

Figure 1: Summary of the origin of some natural products 7

28Figure 2: Leonotis leonorus (L) R.BR

Figure 3: Structure of some compounds obtained from Leonotis ssp... 29

Figure 4: Extraction of active chemical constituents by fractionation 39

Figure 5: Extraction with soxhlet extractor 44

Figure 6: Column chromatography 45

Figure 7: Purification method of active compounds.. 47

Figure 8: TLC of different compounds obtained from methanol extract 54

Figure 9: IR spectrum of P1 65

Figure 10: 1H NMR spectrum of P1.. 66

Figure 11: Mass spectrum of P1 67

Figure 12: IR spectrum of P2 68

Figure 13: 1 H NMR spectrum of P2. 69

Figure 14: Mass spectrum of P2.. 70

XIV

LIST OF ABBREVIATIONS AND SYMBOLS

ADP: Adenosine diphosphate

ATP: Adenosine triphosphate

B.G: Before Christ

CC: Column chromatography

CoA: Coenzyme A

DCM: Dichloromethane

EtOAc: Ethyl acetate

(g): Gas

GABA: Gamma amino- butyric acid

GC/MS: Coupled Gas Chromatography- Mass Spectroscopy

Hex: Hexane

1H NMR: Proton nuclear magnetic resonance

hv: Energy

ip: Intrapertoneally injected

IR: Infrared

(I): Liquid

M5: Mass spectrometry

NADPH: Nicotinamide adenine dinucleotide phosphate (reduced)

N M DA: N-methyl-D-aspartate

PLC: Preparative layer chromatography

SEM: Standard Error mean

TLC: Thin layer chromatography

UV: Ultraviolet

1

Chapter 1

INTRODUCTION

2

Introduction

Natural product chemistry is a science, which studies different products from

living matter, animals or plants. These products are very important for

mankind. According to their nature, natural products can be used for different

purposes. For instance, humans use them for pharmaceutical purpose, for

preparing foodstuffs, insecticides, antioxidants, colouring matters, flavours

and fragrances, extraction of enzymes, pheromones and so on (Marquis,

1981; Torssell, 1983; Cox, 1990; Brunetton, 1999).

Considering the importance of natural products, humans began to do

research on them a long time ago. For instance, Friederich W. Serturner

obtained morphine from opium in 1806; camphor was obtained by Bouchardat

in 1845, cocaine by Niemann in 1859 and so on (Manitto et al., 1981).

Because of their chemical complexity and important physiological properties,

their study continues to this day. The study of these products has contributed

to the current development of organic chemistry. Indeed, because of their

large structural variety, organic chemists are working on them to widen and

deepen their knowledge of organic reactions and, in particular, they can be

used to verify certain mechanisms known in organic chemistry such as

conformation study ofanalysis, process of molecular rearrangements,

molecular structures, absorption spectroscopy, and many more (Manitto et al

1981)

Many studies on natural products showed that a vast array of these occurs in

the plant kingdom. Thus, the continued use of plants as food, as a source of

beverages and medicines depends on the knowledge of the chemical

constituents that are present. Even though plants are widely used for different

necessities, it should be noted that they are sometimes toxic (Ross et ai,

1977). The toxicity may be due to the different chemicals in the plants, even if

present in small quantities.

3

isolation and the study of each product could help to identify the

compound, which is actually responsible for that property. In this way, the

active and less toxic compounds can be purified and used without any risk.

However, some drugs could possess many compounds which act together to

contribute to the effects of the drugs. Similar phenomena apply to medicinal

plants where the various chemical constituents may have additive or

synergistic effects (Ross et al., 1977)

Nowadays, many organic products from the plant kingdom are studied,

isolated and classified for different aims including medicine. This area of study

is described as phytochemistry, which is the chemical study of plants. Our

study includes phytochemistry and together with the pharmacological aspect,

it served to investigate the activity of the compounds isolated from Leonotis

leonorus. It demonstrates the relationship between anticonvulsant activity and

chemical properties of the active agents. Among the plants used in South

Africa for treating epilepsy, Leonotis leonorus (L) R.aR. has been chosen for

investigation in our project. This is due to the following reasons: it is widely

found in South Africa and distributed from the South-western Cape to Eastern

Cape. It is among the plant medicines most widely used by the South African

traditional manage or control ailments,medicine practitioners to cure,

According to Watt et al. (1962), the leaves of Leonotis leonorus are smoked

for the relief of epilepsy. This could be in conformity with the organic

compounds found in the plant,

In spite of its high utilisation in South African traditional medicine, few studies

have been done on this plant. The traditional medicine practitioners use it

without knowledge of the active agents present and/or its mechanism of

action. However, few studies done on the plant show that it possesses

anticonvulsant activity. According to Bienvenu et al. (2002), aqueous extract

of this plant contains anticonvulsant activity. The same authors also studied

the possible mechanism of the crude aqueous extract of the plant and

4

found that this may be a non-specific mechanism, since it affects both

gabaergic and glutaminergic systems. Phytochemical studies carried out on

the plant shows that it contains tannins, quinones, saponines, alkaloids and

triterpene steroids (Laonigro et al., 1979; Bienvenu et alo, 2002).

Thus our study is principally to isolate, purify and pharmacologically evaluate

the active chemical constituents responsible for the anticonvulsant activity of

Leonotis leonorus (L) R.BR.

6

2.1. General view on traditional medicine

2.1.1. Definitions

2.1.1.1. Traditional medicine

According to Sofowora (1982), traditional medicine can be described as a

total combination of knowledge and practice whether explicable or not, used

in diagnosing, preventing or eliminating a physical, mental or social disease. It

may rely exclusively on the past experience and observation handed down

from generation to generation verbally or in writing

Among the material used in traditional medicine, plants playa major role.

According to Marquis (1981) and Sofowora (1982), plant medicine is one of

the oldest practised by mankind. For instance, in 1500 BC, the seeds of the

opium poppy (Papaver somniferum L.) and castor oil seed (Ricinus communis

L.) were excavated from some ancient Egyptian tombs, which indicates their

use at that time. Furthermore, the efficacy of chaulmoogra oils from species of

Hydnocarpus gaern used in the treatment of leprosy was recorded in the

Pharmacopoeia of the Emperor Shen Nung of China between 2730 and 3000

before Christ.

2.1.1.2. Medicinal plants

A medicinal plant is any plant which, in one or more of its parts, contains

substances that can be used for therapeutic purposes or which are precursors

for the synthesis of useful drugs (Sofowora, 1982). Thus, its importance in

medicine is based on organic compounds that possess pharmacological

properties. Nevertheless, these products are always found together with many

other compounds with are often toxic. Identification of non- toxic medicinal

plants and resolution of the problem of toxicity with medicinal plants can be

7

realized through proper scientific investigations

traditional medicine practitioners continue to

In fact, to treat diseases,

use plants without any

knowledge of their chemical composition and their toxicity profile,

2.1.2 Advantages and disadvantages of traditional medicine

Even though plant medicine has been in use for a long time it however has

several disadvantages among which are:

-The lack of hygiene that can cause diseases from microbes and impurities

such as pesticides or other chemicals which can be sprayed on the vegetation

by automobile, agriculture methods, and so on (Sofowora, 1982; Pearson,

1995).

The lack of scientific proof of its efficacy and toxicity profile (Sofowora,

1982). Toxic chemicals, such as selenium and arsenic are naturally present in

some soils and can contaminate medicinal plants growing in such areas

(Pearson, 1995). Such contaminated plants unknown to traditional medicine

practitioners could be used in patients resulting in other serious health

problems.

-The insufficiency and often the imprecision of diagnosis done by

practitioners before giving drugs. Such imprecision is due to the fact that the

traditional medicine practitioners do not know the pathology of certain

diseases. In this case, the traditional practitioners treat the symptoms rather

than the disease, which can some times lead to further complications

(Sofowora, 1982).

Despite the above listed disadvantages, traditional medicine also has many

advantages, which can be exploited for their improvement. Medicinal plants

are potential sources of new drugs, sources of cheap starting materials for

synthesis of known drugs or a cheap source of known drugs. The drugs from

natural sources are better accepted by the body than substances invented in

the laboratory.

8

It has also been noted that traditional medicine is cheaper and more easily

available (Sofowora, 1982; Pearson, 1995). The efficacy of medicinal plants

has encouraged chemists and pharmacists to carry out rigorous analysis on

the plants and to establish a relationship between chemical composition and

therapeutic activities. However, this area of research is far from being

exhausted. It is important to note that many plant constituents remain without

chemical structures (Dimayuga et al., 1991). Nevertheless, several interesting

compounds from plants have been discovered as at date. For instance, in

modern medicine there are several drugs whose origins many people are

unaware of. Obvious examples are mentioned in table 1 (Vickery et al., 1979;

Trease et al., 1983). Besides drugs, other important products, which can be

used in the production of materials used in medicine or in different industries

have been discovered from plants (Vickery et al., 1979; Charchat et al., 1997).

9

Table1: Some examples of drugs from plant kingdom

Name of drug Name of plant Therapeutic group

Digitoxin Digitalis lanata Cardiotonic

Vinca mine Catharanthus lamoeis Antihypertensive

Theobroma cacaoTheophilline Diuretic / Asthma

Ergometrine Claviceps purpurea Oxytocics

Ephedrine Ephedra spp. CNS stimulant

Pilocarpine Pilocarpus spp Cholinergic

Emetine Cephaelis Antiprotozoal

ipecacuanha

Catharanthus roseusVinblastine Antineoplastic,

antileukaemia

Vincristine Antineoplastic,Catharanthus lanceus

antileukaemia

Quinine Cinchona spp Antiprotozoal / malaria

Atropine Datura spp Mydriatic,

antispasmodic

PhysostigmaPhysostigmine Cholinergic

venenosum

Hyoscine Datura spp Sedative!

anticholinergic

Reserpine Rauvolfia vomitoria Hypertension

Conessine Holarrhena floribunda Antiamoebic dysentery,

anthelmintic

Treatment ofStrophanthidin Digitalis purpurea

heart disease

Taxus brevifolius~

AnticancerTaxal

10

2.2. Natural products

2.2.1. General and definitions

As stated earlier, the various chemical constituents in a medicinal plant may

all together contribute to its efficacy These chemical compounds are very

diversified and represent all functional groups in organic chemistry. These

include hydrocarbons, alcohols, aldehydes, ketones, acids, esters, phenols,

ether phenolic, ethers and so on. These fine and bulky compounds belong to

large groups of compounds known as natural products.

According to Killop

1970),

natural products may be defined simply as any

chemicals, which are produced by living matter, and humans have utilized

such compounds since the beginning of time. These products mayor may not

be used by plants or animals for their existence (Manitto et al., 1981;

Koskinen, 1995). Polysaccharides, proteins, fats and nucleic acids, which are

required by living matter as fundamental building blocks, are considered as

the primary metabolites. The whole range of processes by which organisms

synthesize and ~tilise these substances, in order to survive, constitute the

primary metabolic processes. Other chemical processes take place only in

certain species or give rise to different products according to the species.

Such reactions do not appear to be essential for the existence of the organism

and hence are called secondary metabolic processes,

2.2.2. Classification of natural products

Natural products are extremely varied and very complex. Therefore, their

classification is often difficult. However, after much research done on these

chemicals, the classification has been based according to their chemical

structures, biological origin, taxonomic origin or physiological activities

(Torssell, 1983; Trease et al., 1983). Each classification has been

independent of each other. The chemical structure classification is done

according to molecular skeleton and there are four principal groups such as

compounds with linearaliphatic chains, cyclic compounds, aromatic

compounds and heterocyclic compounds.

The classification based on physiological activity gives rise to many groups of

compounds. Each group has a common characteristic based on biogenetic

origin or on chemical functions, which can be responsible for the said activity.

For instance, the cardiotonic compounds are characterised by the lactone

group fixed at C17 of the steroidal ring system, the sterols possess an

alcoholic group at C3 and two methyl groups respectively at C1o and C13 and

so on (Ross et al., 1977). The taxonomic classification is done on the basis of

botanical origin. The biogenesis classification is done according to precursor

products (Ross et al., 1977; Trease et al., 1983). Following the combination of

the different methods of classification, the following principal groups of

compounds could be obtained:

2.2.2.1. Carbohydrates

.These are a group of compounds comprising all varieties of sugars. They are

formed from glucose, which is derived from the reaction between carbon

dioxide and water in plant photosynthesis. After several bioorganic reactions,

glucose gives rise to other simple carbohydrates and these can be combined

to make more complex polysaccharides. According to the number of carbon

atoms or the number of units in the molecule many groups of carbohydrates

are noted: trioses, pentoses, hexoses, monosaccharides, disaccharides,

polysaccharides and so on. Sugars containing an aldehyde function are

termed aldoses and if they contain a keto function, ketoses.

The carbohydrates are very important in living matter where they playa key

role in primary biosynthesis, production and storage of matter and energy

(Ross et al., 1977; Torssell, 1983).

12

2.2.2.2 Glycosides

According to Koskinen (1995), glycosides are formed from the combination of

simple carbohydrates and other products. In this way, they always contain a

saccharide unit attached to a non-carbohydrate moiety which is termed the

aglycone. The term glycoside is generally used if the sugar unit is glucose.

However, other terms can be used for instance, fructoside can be used in the

case of fructose. Also included in this group, are the saponins in which the

aglycones are triterpenoid or steroidal (Ross et al., 1977).

2.2.2.3 Terpenoids

They are compounds, which are typically derived from the isoprene unit. This

group includes different aromatic compounds, vitamins and steroids. The list

below includes terpenoids arranged according to the number of isoprene units

that make up the molecular structure: hemiterpenes (1 unit), monoterpenes (2

units), sesquiterpenes (3 units), diterpenes (4 units), sesterpenes (5 units),

triterpenes (6 units), carotenes (8 units), and polyisoprenes (n units) (Manitto.et al., 1981 and Torssell, 1983).

2.2.2.4 Alkaloids

The term alkaloid was originally coined by Meissner in 1818 from the term

alkalis which means basic compounds. Alkali was derived from Arabic word

"al qalay", which means to roast (Koskinen, 1995). However, it was observed

that not all alkaloids are basic in character and this lead to a new definition,

They are generally defined as substances which contain one or more nitrogen

atoms usually in combination as a part of a cyclic system (Harborne, 1984). It

is noted that this definition also has its shortcomings and must be carefully

used. In fact not all nitrogen-containing compounds are classified as alkaloids

For instance, amino acids, polypeptides and proteins all contain nitrogen but

13

are not considered to be alkaloids. Alkaloids are often toxic to humans and

many have physiological activities, hence their wide use in med1cine

(Harborne, 1984). Various classes of alkaloids exist depending on the ring

system that is present (Koskinen, 1995)

2.2.2.5 Flavonoids

The flavonoids are biochemically formed from shikimates and polyketides

They form the largest group of oxygen heterocycles found in plants and their

structures vary very widely (Koskinen, 1995). According to their common

structures, they are compounds possessing 15 atoms of carbon and two

benzene rings joined by linear three-carbon chain. They are thus,

diarylpropanes. Included in this class of compounds are isoflavonoids ( .

diarylpropanes) and neoflavonoides (

13-,

1.2-

,1,1- diarylpropanes). All these classes

are derived from the most common group of compounds, flavones, which

possess an oxygen bridge between the ortho position of the first ring of

benzene and the benzylic carbon atom adjacent to the second ring (Manitto et

al., 1981). The term flavones is derived from the Greek word, flavius which.means yellow, the characteristic colour of these compounds (Koskinen, 1995).

2.2.2.6 Coumarins

These types of compounds are characterised by the benzo-2-pyrone nucleus.

They are formed from shikimic acid through cinnamic acids via ortho-

hyd roxylation , trans-cis isomerisation of the side chain double bond, and

4

2.2.2.7 Fatty acids and fats

These are compounds soluble in organic solvents. They are biosynthesised

from acetyl CoA through polyketides. The lipids group of compounds, which

are fats, are formed from glycerol-3-phosphate and fatty acid CoA and include

waxes, phosphoglycerides and also other hydrocarbons of quite different

biogenesis such as steroids

Torssell,

1983)

2.2.2.8. Pep tides and proteins

These are from polycondensation of amino acids which are joined by an

amide bond or a peptide bond. This is formed after joining of the carbonyl

group and the amino group respectively of the first and the second amino

acids. The two free functional groups obtained are able to undergo further

condensation processes and thus chain extension is possible. Peptides are

usually polymers of molecular weight lower than about 5000 whereas the

molecular weight of proteins ranges from about 5000 to several millions

'Torssell, 1983).

2.2.2.9 Tannins

The tannins are formed from molecules of phenolic acids such as gallic and

ellagic acids which are joined by ester linkages to a central glucose residue.

Tannins are natural substances with a molecular weight of between 200 and

3000. They possess free phenolic hydroxyls allowing the formation of stable

cross-linkage with proteins and other biopolymers such as cellulose and

pectins. These compounds are considered as excretion products of many

plants but are probably involved in defence mechanism against parasites and

grazing animals (Manitto et al., 1981;Torssell, 1983; Harborne, 1984). For this

reason gallic acid, for example, can be combined with proteins to produce a

deterrent effect on herbivores (Fricks, 2001).

15

2.2.2.10 Resins

These are amorphous substances consisting of the hardened secretions of

plants. They are usually hard, brittle solids, which soften and fuse on heating.

They are soluble in organic solvents but not in water and may generally be

volatile oils (Ross et al., 1977; Springboard, 2000).

It is pertinent to mention here that this represents a brief overview of some

natural products and it is thus necessary to state that many more products or

compounds exist which space does not allow to be discussed

2.2.3. Summary of natural products biogenesis

Natural products are formed from green plants which can convert light energy,

from the sun, into chemical energy (Trease et al., 1983; Brunetton, 1999).

This phenomenon is known as photosynthesis. It takes place in chloroplasts,

cytoplasmic organelles rich in chlorophyll. The light energy absorbed by plant

contributes to th~ production of A TP from ADP and Pi:

ADP + Pi hv ATP

A TP is the coenzyme and the high energy of its terminal phosphate bond is

available to the organism for the supply of the energy necessary for

endergonic reactions. The sunlight energy is also required by water photolysis

and NADP reduction:

2 NADPH + 02+ 2 HNADP + 2 H2O hv

Later, the A TP and NADPH formed are used by green plants for synthesising

16

Thus the basic overall reaction of photosynthesis process is as follows

6 CO2 (g) + 6 H2O (I) hv=700nm C6H1206 (solution) +6 02 (g)

this reaction is a summary of several other processesHowever,

mentioned in the present thesis (Manitto et al., 1981; Trease et al., 1983).

other elements, which form part of the composition of natural products, such

as nitrogen, are minerals and their concentration changes according to the

soil composition in which the plants grow.

From the glucose produced, several other synthetic reactions catalysed by

enzymes (biosynthesis) occur and provide all classes of organic products

found in plants (Figure 1). The basis of natural products in animals, come

from green plants, which serve as food for them

18

2.2.4. The accumulation factors of natural products in plants

Production of natural products by plants is dependent on several factors

which include plant species, ecological factors (season, source and so on),

the plant part to be studied and tne time of plant development (Manitto et ai,

1981; Trease et al.,1983; Torssell, 1983; Lipp, 1988; POR, 2000).

According to Lamaty et al. (1995) and Chalchat et al

1997)

the concentration

of one compound changes according to the species. In fact, the quantity of

eucalyptol at the Ruhande arboretum in Rwanda is the 71.2%, 55.4%, 39.6%,

18%, and the 0.47% for Eucalyptus globulus subsp globulus, Eucalyptus

citriodora hook, Eucalyptus patens benth, Eucalyptus goniocalyx f. muell and

Eucalyptus dives schauer oils respectively. On the other hand, according to

Torssell (1983), some products can change with season. For instance, young

oak leaves contain very little tannin but the concentration increases during the

summer season to reach a maximum in the autumn. Furthermore the solanine

concentration in potato leaves is reduced during the growing season. The

development of plants or of their parts can contribute to the variation in the

natural products found in them. According to Sang-Soo Kwak et al. (1995),

the concentration of taxol during the seed maturation changes and reaches a

maximum level during the middle stage of the seed development and then

decreases with further maturation. The same author showed that the parts of

plant studied must be determined. In fact, according to his study, the embryo

tissue and whole seed had the highest taxollevel when compared with that of

the endosperm and testa. Others authors have shown the influences of other

factors on the production of natural products For instance, according to

Penfold (1951). the concentration of citronellol in Eucalyptus citriodora oil is

56.2%, 80.5%, 79.8% and 73% respectively for Australia, Java, Guatemala,

and Porto-Rico. It changes according to the countries, which have vastly

different ecological elements. According to Tome et al. (1995), daylight and

19

temperature can change the concentration of some natural compounds, for

example, alkaloids,

2.2.5. Utilization of natural products

Natural products are very important to human beings. According to van Wyk

et al. (2000), the active ingredients in medicinal plants are chemical

compounds that act directly or indirectly to prevent or treat disease and

maintain health, This justifies their wide use in medicine, The research done

on these products helped in the discovery of many compounds with diversified

activity. Besides their application in medicine, they can also be used in

different areas such as organic synthesis, in cosmetic, in flavouring agents,

insecticides and in the food industry (Vickery et al., 1979; Marquis, 1981).

2.2.5.1 Natural products and medicine

Before the advent of synthetic organic chemistry natural products were the

sole source of, experimental medicinal materials (Bulger, 1983). The

pharmacologist used the crude extract for treating diseases. With further

advances in technology, the purification, structural studies and synthesis have

Thus, many chemicals used inbeen carried out from natural products.

medicine derived from plants are now available (Table 1 According to Cox

(1990), the importance of plant-derived pharmaceuticals can be deduced from

their prominence in the market. For instance, the Farnsworth's 1984 analysis

of National prescription Audit of 1976 revealed that 25% of all prescriptions

issued in the United States and Canada contained an active component

derived or originally isolated from higher plants. Van Wyk et al. (2000) have

also cited the high use of natural products in medicine. According to them,

natural products and their derivatives represent more than 50% of all drugs in

clinical use in the world

20

2.2.5.2 Natural products and their use in synthesis, cosmetic,

flavour, and food industries

Besides their use in medicine, natural products are also widely used in other

areas. They can be used in organic chemistry for the synthesis of many

compounds with diversified uses. For example, citral from Cymbopogon

citratus can be converted to p-ionone, which is also important as the starting

material in the commercial production of vitamin A1. Conessine from

Holarrhena floribunda is used as a starting material for the commercial

synthesis of some hormones. Sarmentogenin is important as a starting

material for the manufacture of the drugs cortisone and its derivatives, which

are used in the treatment of rheumatoid arthritis (Vickery et al., 1979).

Diosgenin from Discorea floribunda can be used as the starting material in the

commercial synthesis of cortisone and its derivatives and also for

synthesis of hormones used in oral contraceptives (Vickery et al 1979; Cox,

1990). Hydrogenation of piperitone or hydrochloration, acetylation, reduction

and oxidation of Q- phellandrene gives rise to the hemisynthesis of menthol,

which is used in the food and cosmetic industries (Chalchat et al., 1997). On.the other hand, natural products can be used in several industries for

manufacturing insecticides, odorant products and so on. In line with this, citral

from Cymbopogon citratus, limonene and nerol from citrus species, octan-2-01

from Pe/argonium graveo/ens are used in perfumery, the pyrethrins from

Chrysanthemum cinerariaefo/ium, are used as highly effective non-toxic

insecticides. Quinine from cinchona species was used to give a bitter flavour

to soft drinks such as tonic water. Furthermore, herbs and spices have been

used to improve the flavour of foods according to the natural compounds they

contained (Vickery et al., 1979). It should also be noted that natural products

can sometimes be used by plants themselves for different purposes. For

instance, according to Ma.nitto et al., (1981), secondary metabolites often play

a key role in the survival of species over others: defence chemicals, sex

attractants, pheromones etc

21

For example, the alkaloids, because of their biological activity and bitter taste,

may constitute part of the plant's defence mechanism thus, helping to

minimise attack on the plant by animals and insects. In addition, according to

Torssell (1983), the increasing concentration of tannin in some species of

tree serves as protection against different species of animals

2.2.6. Isolation of natural compounds

2.2.6.1. Overview

Before embarking on the study of natural products it is important for the

investigator or researcher to have sufficient information on the origin of the

plant materials to be tested. It is also pertinent to ha\fe an idea of the claims of

therapeutic successes of plants by traditional medicine practitioners. To

achieve, this several ways could be used such as consulting scientific

publications, enquiring from villagers about plants growing near them,

consulting herbalists, and many more (Sofowora, 1982). The collection of

such information enables the investigator to know exactly which part of the.plant could be collected and studied for the research programme.

The collection of plant material must be only from pl.3nts which are free of any

disease and not be affected by viral, bacterial, or fungal infection. In a

diseased plant, not only may products of microbial synthesis be detected in

such plants, but infection may also seriously altE~r plant metabolism and

unexpected products could be formed possibly in large amounts. According to

Lipp (1988), to obtain the best results of chemical analysis from the plants

collected, it is important to handle them properly, s;eeing that the plants are

not mixed with some other materials of similar appearance. For this reason,

an expert in plant identification must authenticate thE~ plants when collected.

22

2.2.6.2 Extraction of natural products

After collection, the plant material must be prepared before extraction can

proceed. The preparation of plant material must be done very carefully to

avoid using both plant and impurities for the extraction of active agents. For

this reason, the washing of plant material with distilled water, immediately

after its collection is very necessary (Navarro et al., 1995; Amabeoku et al.,

2000). Depending on the type of compound needed, the extraction could be

done with fresh or dried material. If drying of plant material is needed, it must

be carried out under controlled conditions to avoid too many chemical

changes occurring. It should be dried as quickly as possible, without using

high temperatures, preferably with a good air draft (Harborne, 1984).

Many methods could be used for extracting natural products. Each method is

specific for the type of compound being isolated. For example, the Clevenger

apparatus is likely to be used for getting essential oils and the fresh part of the

plant is generally used for this purpose (Ross et al., 1977). However, dried

materials can also be used in certain cases. For instance, the official

monograph for gentian, used as a bitter tonic, stipulates that it should consist

of the dried fermented rhizome and root of Gentiana lutea. Indeed, fresh

gentian has a sweet pleasant taste and only acquires its characteristic

bitterness after fermentation during which the di- and tri-saccharides are

partially converted into monosaccharides. Similarly, Vanilla pod, the source of

natural vanillin, contains the phenolic glycoside, glucovanillic alcohol. During

the curing process, which involves slow drying, the glycoside is enzymically

hydrolysed and oxidised to vanillin (Ross et al., 1977). The fresh plant

material can also be used when fixed oils are needed. For this purpose, the

application of pressure may be necessary. This also applies in the case of the

preparation of castor oil, olive oil, and so on.

23

When extraction is done on dried plant material, a classical chemical

procedure for obtaining organic constituents is to continuously extract

powdered material in soxhlet apparatus with a range of solvents (Harborne

1984). In this way, it is important to know how to choose the solvent to be

used. If the type of compounds being isolated is known, selective solvent

extraction will make the process safer. If not, the usual way is to start with a

non-polar solvent and exhaustively extract the material, followed by a series

of more polar solvents, until several extracts of increasing solute polarity are

obtained. Based to the increasing polarity, the classical solvents are light

petroleum, cyclohexane, toluene, dichloromethane, chloroform, diethyl ether,

ethyl acetate, acetone, n-propanol, ethanol, methanol and water

(Ross et al., 1977; Williamson et al., 1996).

2.2.6.3. Separation of compounds and purification

During extraction with solvents only crude extracts are obtained. These must

be investigated for activity to be studied to determine which of the extracts

possess the best activity. Following the extraction, the separation of,

compounds from plant extracts could be done. It must be done very carefully

and during this procedure, patience is needed. According to Williamson et al.,

(1996), the more precise the isolation procedure is the better, because it is

more accurate to investigate the biological activity of a single compound than

a mixed range which may contain both agonists and antagonists in the same

extract. In the past, it was very difficult to isolate pure compounds by chemical

methods (Killop, 1970). However, nowadays, with the introduction of modern

scientific methods, the process of separation and purification of mixtures of

natural products has been considerably simplified. In addition, it has been

shown that chromatographic techniques playa very important role and it is the

advent of these separation methods that has been primarily responsible for

the rapid progress in natural products chemistry and related disciplines (Ross

et al., 1977).

24

The most important of these methods are: Thin layer chromatography (TLC),

Preparative layer chromatography (PLC), Gas liquid chromatography (GLC),

Column chromatography (CC), Paper chromatography (PC), and High

performance liquid chromatography (HPLC). As for the extraction process, the

polarity of the solvent to be used must be considered. The extracts obtained

need to be concentrated using a rotary vacuum evaporator. Generally, the

more polar the solvent, the more heat is required to evaporate it and that

could decompose the products that are being looked for. Thus, the most

volatile solvent that will be effective is chosen. However, mixtures of them are

often used (Wagner et al., 1984, Sewell et al., 1987). With the

chromatographic methods, many fractions of compounds can be obtained and

tested for biological activity. The pharmacological test must identify which

fractions possess the activity and these can undergo further separations

(Williamson et al., 1996). In fact, once the biologically active fractions are

obtained, further purification could be undertaken to identify the constituents

to find out where the biological activity actually lies.

2.2.7/dentification of natura/ compounds.

Generally, the identification of natural compounds uses both, physical and

chemical methods. However, depending on the purpose of the study, only one

method among them could be used. In the past, the class of compounds was

usually obtained from its response to colour tests, its solubility, retardation

factor (Rf) properties and its UV spectral characteristics. Furthermore, other

characteristics could be verified for confirmation. These include melting point

for solids, boiling point for liquids and optical rotation for optically active

compounds (Harborne, 1984). According to modern methods, equally

informative data on plant substances are its spectral characteristics. The

spectral methods used include infrared (IR) spectra, nuclear magnetic

resonance (NMR) spectra, mass spectra (MS) and so on. According to

Harborne. (1984). the comparison with authentic material. in the case of a

25

known compound, must be done for confirmation. But, if authentic material is

not available, careful comparison with literature data may suffice for that

identification. According to the same author, if a new compound is present

data from previous spectral methods should be sufficient to characterise it,

spectral method provides its own information about the molecular

structure. For instance, the infrared spectrum (IR) gives information on

masses of the atoms, and the forces holding them together. It is most useful

for determining what functional groups are present in a molecule. The nuclear

magnetic resonance (NMR) spectrum gives information about the

environment of each set of hydrogen atoms and determines which carbon

atom is bound to which hydrogen atom and also can show what functional

group or heteroatom is nearby. The mass spectrum (MS) is obtained when a

molecule is subjected to a high-energy electron bombardment under vacuum

and in magnetic field Basic information about the molecular formula and

fragmentation patterns gives structural identity to the molecule (Sorrell, 1988;

Verpoorte, 1989). All these methods are generally used together for easy

identification. In our experiment certain methods from those above have been

used to analyse ,the active components with anticonvulsant activity obtained

from the leaves of Leonotis leonorus.

2.3. Synopsis of Leonotis leonorus (L) R.BR.

2.3.1. Description and classification

Leonotis is a genus frequently found in Africa. About ten species are known

some of which are Leonotis leonorus R.BR., Leonotis dubia Benth and

Leonotis dysophylla Benth and occur in South Africa (Batten, 1986; Hilliard et

al., 1987). The name leonotis is derived from the Greek word "leon" (a lion)

and "ous", "otis" (an ear), according to their flowers, which resemble lion's

ears. The most attractive of the South Africa species is Leonotis leonorus, a

shrub, which is a conspicuous feature of the autumn and early winter

26

landscape. It is widely distributed from the South Western Cape, through the

Eastern Cape, Ciskei, Transkei, and Natal to Eastern Transvaal. It is found

both at the coast and inland, growing in full sun on flats and hill slopes and

often at the forest margins (Adamson et ai, 1950; Barbara, 1975; Batten,

1986). The plant is a many-stemmed shrub, which usually attains a height of

about 2 m. The leaves are short, petiolate, obiong-lanceolate or lanceolate,

about 5-10 cm long, sfightly oblique at the throat, and have the short teeth on

the rim of the leaf. The orange red flowers being grouped in dense clusters

along the stems characterize the genus. The fruit consists of four little nutlets

seated in the base of calyx tube. Naturally it dies after flowering and new

growth starts once more in the spring. Propagation is by means of cuttings,

division of the rootstock or by seed (Adamson, 1950; Hutchinson, 1973).

Leonotis leonorus, R.Br. belongs to the Angiospermae phylum, subphylum of

Dicotyledoneae in Labiatae (Lamiaceae) family (Adamson, 1950; Hutchinson,

1973). The plant has several names among which are minaret flower, cape

hemp, red dagga, klip dagga, rooi dagga, duiwelstabak, duiwelstwak,

koppiesdagga. In South African it is known as wilde dagga in Afrikaans,.Umunyane or imunyamunya in Zulu, lebake in Sotho, umfincafincane in

Xhosa and umhlahlampetu in Shona (Adamson, 1950; Barbara, 1975;

Hutchings et al., 1996; van Wyket al., 2000).

2.3.2. Indications of Leonotis leonorus

This common wild flower has a long history as a medicinal plant. It is used in

South Africa internally or externally for treating different illnesses. An infusion

and decoction of the leaves and stems has been used internally for coughs,

colds, influenza, bronchitis, high blood pressure, headaches and chest

troubles, and for the relief of bronchial asthma. Externally, the decoction has

been applied to treat cardiac asthma, boils, eczema, skin complaints, itching,

27

muscular cramp and haemorrhoids. It has also been used for treating

snakebites (van Wyk et al., 2000; Bienvenu et al., 2002). According to Watt et

al. (1962), the leaves of Leonotis leonorus are smoked for the relief of

epilepsy.

2.3.3 Some compounds isolated from Leonotis spp

The research already done on Leonotis ssp showed that Leonotis leonorus

possesses many organic compounds grouped into tannins, quinones,

saponins, alkaloids, resins and terpenoids (Watt et al., 1962; Laonigro et al.,

1979; Bienvenu et al., 2002). Some of these have been isolated and

chemically identified as marrubiin, possible artefact of premarrubiin during

extraction (Kaplan et al., 1968; Harborne et al., 1995; van Wyk et al., 2000).

Marrubiin has for sometime been described as a glycoside with melting point

of 156-159 °c (Watt et al., 1962). According to Watt et al. (1962), Marloth

isolated a dark green resin from the leaves of Leonotis leonorus. This resin

has been suspected to be responsible for the narcotic property of the plant.

Other terpenoid lactones including Leonotin have been found in Leonotis.ocymifolia (Habtemariam et al., 1994). All this research has shown that

Leonotis ssp is very rich in terpenoids essentially in diterpenoid lactones.

29

Marrubiin Premarrubiin Leonotin

",0,/0"

Figure 3: Structure of some compounds obtained from Leonotis ssp.

(Kaplan et al., 1968; Habtemariam et a/., 1994; Harborne et al., 1995;

van Wyk et al., 2000)

2.4. General view on epilepsy

2.4.1. Definitions

Epilepsy has been in existence since time immemorial. The name epilepsy

was derived from the Greek word "epilambanein" meaning to seize. In 400

B.C., Hippocrates disputed the myth that epilepsy is a supernatural disease of

the brain and should be treated by diet (Delgado et al., 1970; Vida 1975).

However the full clinical descriptions given by him are still used and have led

to a complete definition of epilepsy (Vida, 1975). According to Ashok et al.

(1988), epilepsy is collectively designated to a group of chronic central

nervous system disorders characterised by spontaneous occurrence of

seizures generally associated with the loss of consciousness and body

movements (convulsions). According to the symptom of epilepsy, seizures

30

can be divided into groups and the form that epilepsy takes depends largely

on the part of the brain which has been injured, the extent of the injury and the

amount and kind of turbance produced in the chemistry of the nerve cells.

When generalised, epilepsy is manifested as seizures, accompanied by

general convulsions, chewing motions and loss of consciousness and is

termed grand mal epilepsy. Another type of epilepsy in which there is

generalised hyperactivity involving essentially all parts of the brain, is petit mal

epilepsy which occurs in two forms: petit mal variant and petit mal. Petit mal

variant occurs in slightly older children and is accompanied by mental defects,

impaired and incoordination of movement; whereas petit mal occurs with

increasing age in which the brain becomes more able to cope with injury. It is

generally associated with mental retardation or cerebral palsy (Delgado et al.;

1970; The Encyclopaedia Americana, 1984). However, according to the

medical symptoms and diagnostic data, detailed classification can be given

(Doiske et al.; 1998).

2.4.2. Causes of convulsion

.The type of seizures depends on the site of the focus in the brain, the regions

to which the discharge spreads and the effects of post ictal paralysis of these

regions. Anything that damages nerve cells in the brain can cause epilepsy: a

blow to the head, brain infection, improper nourishment of brain cells due to

metabolic defects or inadequate supplies of blood to the brain, kidney

disease, poisons or brain tumours. Occasional, sudden, excessive, rapid and

local electrical discharges in the grey matter of the brain give rise to

convulsion (Delgado et al., 1970). The introduction of the

electroencephalogram in medicine explains it and demonstrates that epileptic

convulsions are characterised by an excessive discharge of electricity,

apparently from the dendrites of pyramidal cells of cerebral cortex neurons

(Delgado et al., 1970). Further analysis shows that the blockade of

postsynaptic gamma- amino butyric acid (GABA) receptors or an inhibition of

31

GABA synthesis is the principal origin of brain discharge (Delgado et al.,

1970; Klawans, 1979). GABA is the major inhibitory neurotransmitter in the

central nervous system (CNS),

2.4.3. Impact of epilepsy in society

The decision to treat epilepsy should be taken after weighing the

consequences of the disorder on society. In fact, epilepsy does not pose a

high risk to life itself. However, the detrimental effects of epilepsy on the

quality of life increases as society becomes more complex and technological.Thus,

it is the medical scientists' responsibility to see how to cure this disorder

completely and permanently for the best existence of mankind. Epilepsy does

not only have bad consequences on society but also on the families of

sufferers. Indeed families, who have people suffering from epilepsy, usually

have economic and social problems. According to Gacky (1993), some

families of children with epilepsy have poor communication between the

parents and children, and some times, conflict between the parents may

occur. Epilepsy may also adversely affect the academic and work life of the.sufferer. Early onset in children can affect their schooling and eventual

occupational achievement. Onset in adulthood may affect those abilities and

skills needed to perform a job or obtain employment (Dolske et al., 1998).

2.4.4. The pharmacological evaluation of anticonvulsants

According to Vida (1975), experimental evaluation of anticonvulsant drugs is

based on the observations of Fritsch and Ferrier that seizures originate in the

cerebral cortex. This was demonstrated by causing generalised convulsive

seizures in experimental animals by local electrical stimulation and excitation

of certain areas of the cerebral cortex. Later, it was discovered that some

chemical compounds such as pentylenetetrazole, biccuculine, picrotoxin and

so on also induce convulsions. Seizures may also be caused by sensory

32

stimulation and heat (Vida, 1975). With the advent of convulsant agents,

much research has been done to test for anticonvulsant activity of many

agents which can inhibit the seizures induced in animals and therefore could

be used to treat epilepsy. The evaluation of anticonvulsant activity has

generally been done on mice and/or rats. According to Delgado et al. (1970),

the practical methods involve treatment with the potential anticonvulsant

followed by the administration of a convulsing agent, chemical or electrical

With the electrical method, the minimal seizure that is identified by the

occurrence of detectable clonus of facial muscles and rhythmical twitches of

whiskers and ears minimal electroshock test (MET or the supramaximal

stimulus to elicit the maximal seizure (MES) can be done The chemical

methods usually consist of administration of the convulsant agent in doses

ranging from that which causes convulsion to fatal doses, to experimental

animals pre-treated with the potential anticonvulsant compound. The amount

of convulsant agent necessary to provoke seizures is then determined.

33

Chapter 3

DESCRIPTION OF THE PROJECT

34

3.1 Introduction

Drugs used to treat diseases or infections antagonise any pathogenic agents

responsible for the diseases or infections. A central aim underlying the use of

botanical remedies is similar to that of orthodox medicine because those

plants used in medicine are rich in organic compounds as previously

mentioned. However, there is a major difference between medicines obtained

from plants and those made in the laboratory by synthetic techniques since

plant medicines contain a mixture of unknown compounds some or all of

which may contribute to the efficacy of the medicines. Plant medicine could

possess toxic substances, which may be masked by some other components

also found in the plant. However, the activity of these plant medicines could

be decreased by the presence of these inactive substances. On the other

hand, drugs obtained from laboratory syntheses are chemically known and it

is very easy to determine their structure-activity relationships. It is also easier

to study their toxicity profile. Structure-activity relationship studies can also be

carried out on plant medicines especially after the isolation of active agents

and elimination of all undesirable compounds from the medicines..

As stated earlier Leonotis leonorus (L) R.aR. is widely used by traditional

medicine practitioners to treat many diseases including epilepsy. However, no

chemical study has been done on the components of the plant exhibiting the

anticonvulsant activity. This study is intended, therefore, to investigate the

components of the plant exhibiting the anticonvulsant property,

3. 2 Hypothesis and objectives of the project

Traditional medicine practitioners in the country have used Leonotis leonorus

for the treatment of epilepsy for a long time. Bienvenu et al. (2002) have

shown that its leaves really possess anticonvulsant activity. This activity is

due to the presence of unknown chemical products in the aqueous extract

35

used during their experiments. According to Watt et al. (1962) and van Wyk et

al. (2000), the leaves of Leonotis leonorus were smoked for the treatment of

epilepsy while the infusion or decoction has been used against other

symptoms Therefore, the effects of more lipophilic constituents of the plant

against convulsions would be investigated.

The lipophilic compounds are not soluble in cold water, but are in hot water,

which Bienvenu (2002) used during his experiment. If the components with

anticonvulsant property are actually lipophilic, it will be easy to use other

solvents less polar than water for their extraction. Indeed water is known as

one of the more polar solvents used in chemistry. Thus, it extracts the more

polar compounds. The less polar compounds including these lipophilic ones

are extracted using organic solvents. If these are used, then the compounds

extracted by water are eliminated and this could affect the anticonvulsant

activity of the plant. Thus, components isolated from the new crude extract

(organic) may have higher anticonvulsant activity than the crude extract since

they are also to be purified.

.The isolation of active compounds is sometimes accompanied by structure

elucidation. Once found, the structure of the anticonvulsant agents could

indicate the mode of pharmacological action and allow complete synthesis of

novel analogue products. Furthermore, it can unveil other uses of the

products.

In summary our study has the following objectives:

-To verify the anticonvulsant effect of Leonotis leonorus (L) R.aR.

extracts including aqueous and organic solvents.

-To verify the anticonvulsant effect of purified active agents from

Leonotis leonorus extract

To chemically identify the active agents,

36

Chapter 4

METHODOLOGY

37

4.1 Preparation of plant material

4.1.1 Collection

The plant, Leonotis leonorus (L) R.BR., was collected between 9hOO and

11 hOD during the summer months and in the winter months from Belhar and

the Cape Flats Nature Reserve, Bellville, Republic of South Africa.

respectively. The botanist and taxonomist Mr Franz Weitz of Botany

Department, University of the Western Cape, identified the plant and a

Voucher specimen (TRAD 10) was deposited in the University Herbarium.

4.1.2 Drying of plant material

Before being dried, the leaves mixed with young stems were washed with

distilled water to remove all impurities, which could have been brought to the

leaves by wind, birds, insects, automobiles and so on. They were then placed

on clean plates and kept in a ventilated oven at 30 DC for 72 hours. Then

after that, the dried leaves were ground into a powder with warring.commercial laboratory blender (KENWOOD CG100 PK032/AD, KENWOOD

LIMITED, Great Britain) and further milled (mesh size 850 !.1m).

4.1.3 Preparation of extracts

The extraction was performed in the organic chemistry laboratory, University

of the Western Cape. Three solvents were used for the extraction and they

include hexane 96% (Kimix, Chemicals and Laboratory Suppliers, Cape

Town, South Africa), methanol 99.5% (Kimix, Chemical and Laboratory

Suppliers, Cape Town, South Africa) and distilled water.

38

.The organic extracts were prepared using a soxhlet extractor (Figure 5). 40

g of fine powder was extracted with hexane or methanol at 50 DC for 24 h. The

flask contents were concentrated on a rotary evaporator (Rotavapor RE 111,

BUCHI, SWITZERLAND) under reduced pressure at a water bath

temperature of 50 DC. Afterwards, the extracts obtained were kept at room

temperature for all the solvent to evaporate until constant mass was obtained.

Then the extract obtained was kept in a vacuum dessicator containing calcium

chloride to protect it against humidity.

.The aqueous extract was prepared from 40 g of dried powder, which was

soaked in 500 ml of boiling water and was left to cool with stirring on a

magnetic stirrer for 24 h. The mixture obtained was then filtered and the

filtrate was kept. The material left over was subjected to a second and third

extraction using each time fresh solvent (distilled water). All filtrates obtained

(1500 ml) were combined and placed in 4 round bottom flasks, each having a

capacity of 11; which were kept in a fridge (CFC FREE FREEZER, U85360,

New Brunswick SCIENTIFIC) at -83 DC for 5 h to freeze. The flasks contents

were then freeze-dried in a freeze mobile (VIRTIS 12SL, VIRTIS COMPANY.GARDINER, NEWYORK). A dried fine powder was obtained after 72h and the

mass recorded.

After doing the preliminary anticonvulsant tests on mice, the extraction

protocol was changed and involved successive steps of extraction starting

with the less polar solvent (hexane) and ending with the more polar solvent

(distilled water) as shown in figure 4. The material used remained unaltered.

However, in this second procedure the extraction by fractionation required

drying the powder at 30 0 C before using another solvent.

39

~

Extralction with hexane

?

USins~:=Imarc: extract + solvent

concentration and

overall evaporation

drying and extraction

with methanol using

soxhlet extractor

solvent + e)(tractmarc

concentration and

evaporation

drying and extraction

with hot distilled water

+filtration

mlethanol extrac:

filtratemarc

fridge at -83 °c

freeze dried

aqueous extract

Figure 4: Extraction of active chemical constituents by fractionation

40

4.2 Pharmacological tests

4.2.1 Animals

Male albino mice bought from the University of (~ape iTown. South Africa

weighing 18-30 g were used. The animals were kept in Igroups of eight peri

cage and had free access to food and water. Each anim~1 was used for onei

seizure experiment only. rlr

4.2.2 Drugs and chemicals

Sigma

Rorer,

Chemical Co) amd phenobarbitone

South Africa:~ were all dissolved in

physiological saline. Diazepam (Vallium, Roche Products, South Africa) was

dissolved in a minimum amount of polyethylene glyc:ol400 (Fluca, Buchs) and

adjusted to the appropriate volume with physiological saline. The methanol

extract was dissolved in a minimum amount of mE~thanol (0.3 ml) while the

hexane extract and pure products isolated from the methanol extract by PLC.were dissolved in a minimum amount of Tween 80 (0.2 ml) and all adjusted to

the appropriate volume with physiological saline. All drugs and extracts were

injected intraperitoneally in a volume of 1 ml/1 00 9 of mouse. Control animals

received equal volume injections of the appropriatE~ vehicle. Fresh drug and

plant material solutions were prepared on the days of the experiment.

4.2.3 Anticonvulsant activity assessment

anticonvulsant activities of crude aqueous, methanol and hexane extracts as

well as purified fractions of Leonotis leonorus. Mice were used in groups of

eight per dose of drug or extracts. The animals were kept singly in transparent

4

perplex cages (25 cm x 15 cm x 15 cm) for 30 min t:J acclimatize them to their

new environment before drug or extract treatment. Seizures were elicited in

mice with PTZ. Mice were observed for a period! of 30 min following the

administration of PTZ. Seizures manifested as 'wild r~nning, followed by

stunning or clonic convulsion and then tonic com/ulsio~ exhibited by tonic

The latency or onset of tonic convulsion and thehindlimb extension

proportion of animals convulsing were recorded. Animals that did not convulse

within 30 min of observation were regarded as not convulsing,

The experiment was done with increasing doses of PTZ until a minimum

dose, which induced seizures in 100 % of animals u sed and with a reasonable

onset time was reached. This dose was taken as the working dose and used

throughout the experiment.

To test for anticonvulsant activity the indices of measurement are either

significant delay in onset of tonic seizures or si~;Jnificant reduction in the

incidence of seizures (proportion of animals convulsing) or both (Amabeoku et

al., 1993; Amabeoku et al., 1998). The anticonvulsant activity of each of the.crude extracts, isolates or pure products was done by pretreating the animals

with solutions of the plant materials for 15 min and the standard antiepileptic

drugs, phenobarbitone for 10 min and diazepaml for 20 min prior to the

administration of the convulsant agent, PTZ. The pretreatment times were

obtained from preliminary studies in our laboratory. Control experiments

involving the control vehicles such as methanol, Tween 80 and polyethylene

glycol 400, all dissolved in physiological saline were done concurrently with

the test experiments. All experiments were carried Clut between 9 am and

4.30 pm in a quiet laboratory with an ambient temperature of 22 :t 2 aGo

42

4.3 Isolation of active compounds

The isolation of active compounds from the methanol extract was done using

both thin layer chromatography and column chromatography in the organic

chemistry laboratory, Department of Chemistry, Univer~ity of the Western

Cape. Ethyl acetate, dichloromethane and hexarle us~d in the isolation

experiment were previously distilled,

4.3.1 Choice of solvents

A small quantity (0,05 g) of methanol extract was dissolved in 1 ml of

dichloromethane. By means of the spotter (pasteur pipette), a small amount of

methanol extract was spotted at 0.7 cm from the bottom of the plate (silica gel

60 F254, Merck Germany). The plate was left to d~1 and then placed upright

in a tank containing a small quantity of the solvent ~jystem to be studied: viz.,

a mixture of ethyl acetate and hexane. The solvent mixture was allowed to

rise up the thin layer chromatography plate until it was at 1 cm from the top of

plate. The plate was then removed and the level of the solvent mixture front.was marked for the determination of the retardation factor (Rf) for each

compound detected. The plate was left to dry beforl3 the various bands were

identified. Three different strengths of ethyl acetate I hexane mixture: 10 %,

20 % and 30 % by volume were tested

4.3.2 Detection of spots

The spots were detected in visible light and in UV li~}ht at 254 nm. The colour

obtained for each spot was recorded. The distance between the baseline and

the centre of each spot detected, and the distance between the baseline and

the solvent front were recorded for further analysis,

43

4.3.3 Isolation of active compounds

Both thin layer chromatography and column chromatography have been used,

During column chromatography, the stationary phase was silica gel (35-70

and 70-230) and the mobile phase was a mixture of ethyl acetate and hexane

in proportion of 3:7 by volume. Fraction collector (BIG-RAD, Model 2110) was

used to collect the small fractions. The thin layer chromatography was needed

to pool together similar fractions, which contain the same compounds.

4.3.3.1 Preparation of sample to be separated

10 9 of methanol extract was dissolved in methanol. The solution obtained

was mixed with a small quantity of coarse silica gel in a round bottom flask

and the methanol evaporated with a rotary evaporator at 50 0 C. The extract

was pre- adsorbed onto the silica gel. Then the mixture obtained was kept for

further analysis.

4.3.3.2 Column chromatography

The mixture (sample) previously obtained was fractionated over silica gel (70-

230) column chromatography (Figure 6) and was eluted with a mixture of ethyl

acetate: hexane (3:7) followed by 100 % of ethyl acetate to elute the highly

polar compounds. The collector was used to collect the small fractions and

switched on when the first drop of compound started to flow. The fractions

obtained were analysed by thin layer chromatography and UV light. Similar

fractions were combined in the same flask and concentrated on a rotary

evaporator. The small solvent that remained in the compound obtained was

evaporated at room temperature The quantity of each compound obtained

was recorded

44

Figure 5: Extraction with Soxhlet extractor

45

Figure 6: Column Chromatography

46

4.4 Purification of components with anticonvulsant activity

Column chromatography (CC) and preparative layer chromatography (PLC)

were used to purify the compounds with anticonvulsant activity. Column

chromatography was used for separating major bands of compounds, using a

mobile phase starting with benzene for the first active compound and ending

with EtOAc: Hex (3:7) which eluted the second active compound. The

products obtained were then purified again using large plates of fine silica gel.

The mobile phase used was a mixture of ethyl acetate:hexane (3:7). The

method of plating allowed several small impurities which were eluted together

with the principal products during column chromatography to be removed. A

dark green product was obtained for compound "1 (P1) and passed again

through column chromatography before bein~1 tested on mice for

anticonvulsant activity.

A green yellow compound was obtained for the second compound (P2). P1

and P2 were washed with distilled water to dissolv'e all mineral compounds

that could be present (Figure 7),

47

Active fractions

.CC (Silica gel)

Mobile phases:- Ben:zene I

-EtOAc:HJx (3:7)

.PLC (Silica gel) I:

Mobile phase: EtOAc:Hex (3:7)

Scraping

Impurities

Major component

Dissolution in DCM

Filtration

Filtrate marc

Evaporation at 50°C

Residue

.cc

Figure 7: Purification method of active compounds

48

4.5 Characterisation of active compounds

4.5.1 Characterisation using co/oured react/fans

All the groups of compounds cited to be present in the crude aqueous extract

of the leaves of Leonotis leonorus (Laonigro et al., 19V9; Bienvenu et al.,

2002) have been chemically tested. For this purpose, standard chemical

methods for identification, such as Carr Price test, Anisaldehyde/H2SO4,

FeCI3. Borntrager reaction, Wilstatter reaction, Mayer reagent and Dragendorff

reagent were used for testing the nature of the compounps (Sofowora, 1982;II

Harborne, 1984; Wagner et al., 1984). "#;31

4.5.1. 1 Test of terpenoids

Terpenoids have been tested using anisaldehyde- :sulphuric acid reagent. To

prepare this reagent, 0.5 ml of anisaldehyde was mixed with 10 ml of glacial

acetic acid and 85 ml of methanol followed by 5 ml of concentrated sulphuric

acid was added to the mixture. Then three drops of anisaldehyde- sulphuric.acid reagent were added to the test solution. The appearance of a blue,

reddish green or brown coloration confirms thE~ presence of terpenoid

compounds (Wagner et al., 1984). c:,!t.

4.5. 1.2 Test af sapanins

A presence of saponins is confirmed by reddish violE~t colour following addition

of 2 to 3 drops of Carr Price reagent (5 % SbCI3 in chloroform) to the test

solution (Harborne, 1984). However, in this stuldy, no such colour was

obtained with our test solution.

49

4.5.1.3 Test of tannins

For the test of the presence of tannins, iron (III) chlori,de reagent (5 % in

distilled water) was used. The presence of tannins was indicated by the

appearance of blue- black precipitate following thE! addition of few drops of

reagent in the solution (Harborne, 1984; Wagner et al 1984)

4.5.1.4 Test of qui nones

Borntrager reagent (5 % of KOH in ethanol) has been used to test for the

presence of quinones in the solution. A small volume of HCI 10 % was added

to the solution to be tested. The mixture obtained ,,,,as 1~ft to stand for 12 h.

Then, the Borntrager reagent was added. An appearance of a red colour

indicated the presence of quinones in the solution I (Santesson, 1970;

Harborne, 1984)

4.5.1.5 Test of alkaloids

Dragendorff reagent and Mayer reagent were used to testlfor alkaloids.

Dragendorff reagent was prepared by the dissollJtion lof 0.85 g of basic

bismuth nitrate in 40 ml of distilled water and 10 ml of glacial acetic acid.Then,

8 g of potassium iodine dissolved in 20 ml of distilled water was added

to the mixture. Mayer reagent was prepared by dis~)olution of HgCI2 in 30 ml

of distilled water and the obtained solution was added to 2,5 9 of potassium

iodine dissolved in 5 ml of distilled water. The solution was adjusted to 50 ml

using distilled water. These two reagents were prE~pared on the day of the

The presence of alkaloids was indicated b~ the appearance ofexperiment.

precipitate following the addition of few drops of Dra!~end9rff reagent or Mayer

reagent to the solution (Wagner, 1984). ri

50

4.5.1.6 Testofflavonoids

Flavonoids could be reduced by HCI in the presence of Mg or Zn (Wilstatter

reaction) to give coloured substances, anthocyanins (Manitto et al., 1981;

Sofowora, 1982). This reaction was done to test thE~ pres~nce of flavonoids in

the solution. Few drops of the solution containing HCI:CH~OH:H20 (1:1:1) and

a small quantity of Mg were added to the test solutionf The colour of red,

orange or violet indicates the presence of flavonoids. However, no such

colour was obtained in this study.

4.5.2 Characterization by spectroscopic methods

For this purpose IR, 1H NMR and GC/MS were uselj. The IR absorption of P1

and P2 in KBr has been studied using Perkin Elmer PARAGON 1000 PC FT -

IR Spectrophotometer. The mass spectra of these compounds have been

obtained with coupled GC/MS spectrometer: Finni~~an Mat GCQ GC / Mass

Spectrometer, wrile 1H NMR spectra were obtained usi,ng a Varian Gemini

XR 200 NMR Spectrometer.

4.6 Pharmacological results analysis

The results from the anticonvulsant assessments 'Nere analysed using both

paired Student's t- test and chi- squared test for the onset of seizures and the

proportion of animals that exhibited tonic convulsion respectively (Tallarida et

al., 1981)

52

Res u Its

In this chapter symbols have been used to represent different extracts

fractions and pure compounds obtained from plant material. HE indicates the

hexane extract while ME1 and ME3 represent the metha~ol extracts obtained

from the plant material collected during the summer months at Cape Flats

Nature Reserve and Belhar respectively. ME2 indic:ates the methanol extract

obtained from the plant material collected at Cape Flats Nature Reserve

during the winter month. AQ and AQM represent the aqueous extracts

obtained from the original fine powder and methano~ extraction residue

respectively. F1. F21 F3. F4 and Fs indicate the different fractions obtained from

methanol extracts (ME11 ME3) while P1 and P2 represent T o pure compounds

obtained from F1 and F2 respectively using CC and PLC."

5.1 Extracts obtained from fine powder

The preliminary extraction done on separate 40 g quantities of fine powder

using three diff~rent solvents gave yellow, greenl and! brown extracts for

hexane, methanol and water respectively. The quantity of the crude extracts

after evaporation of the solvents were 2.82 g, 10.8 g and113.50 g for hexane

(HE), methanol (ME) and aqueous extracts (AQ) re:5pectively. The respective

mean yields of 7.05 %, 27.00 % and 33.75 % were calculated from the fine

powder.

5.2 Yield obtained after extraction by fractionation

Sequential extraction of a sample of 890 g of the powder by firstly hexane,

then methanol and finally water gave 64.5 g, 220 ~) and 159.18 g, with mean

yields of 7.24 %, 24.72 % and 6.64 % for hexane (HE), methanol (ME

)

and for

aqueous (AOM) extracts respectively. The methanol extracts was difficult to

dry and contained some solvent.

53

5.3 Characterisation of compounds obtained from methanol extract

(ME1, ME3)

Five fractions were obtained from methanol extract using both CC and TLC.

These groups of compounds have been characterised I by their retardationI

factors (Rf) and their colours visible to the naked eye on ~LC plate and using

UV light at 254 nm. Compound F1 was seen as dark blule and characterised

by its retardation factor of 0.80. Observed under U\' light~ it was dark.

Compound F2 was seen as greenish yellow and c~aracterised by its

retardation factor of 0.66. It was also coloured as reddish brown under UV

light. Compound F3 with retardation factor of 0.45 \"v'as seen as green on TLC

and was also characterised by a red colour under UV light while compound F4

with retardation factor of 0.32 had a yellow colour on TyC and was seen as

reddish brown under UV light. The last compound F;s found was dark blue on

TLC and red under UV light. Its retardation factor 'was evaluated to be 0.06

(Table 2).

.Table 2: Characteristics of compounds found from nnetharol extracts

(ME11 ME3) I; 1r

Zone Symbol of compounds Retardation factor (Rf) Colour (Vision)

1 F1 0.80 Blue dark

2 F2 0.66 Yellow greenish

3 F3 0.45 Green

4 F4 0.32 Yellow

5 Fs 0.06 Blue dark

54

Mixture of compound in methanol extract.

Compounds obtained from CC

A B c

A. Five fractions obtained

B. Compounds P1 and P2 before purification

C. Compounds P1 and P2 after purification

Figure 8: TLC of different compounds obtained from methanol extract

55

5.4 Yield of anticonvulsant agents

The fractionation of methanol extract obtained from ,890 glof fine powder gave

two active compounds P1 and P2. After their purifi(;ation using both CC and

PLC the quantity obtained for P1 and P2 were 2.55 ~~ and 1.60 g, respectively.

The mean yield may be expressed as 0.28 % for P1 and °r18 % for P2.

5.5 Chemical identification from test tube reactions

Three inactive fractions F3, F4 and Fs and purified compounds P1 and P2 were

tested so as to have complete information on chemical composition of

methanol extract. F3 gave the positive test with anisaldehyde/ H2SO4 reagent

and with Borntrager reaction which indicate the presence of terpenoid and

quinones respectively. F4 gave positive test witlh both Dragendorff and

Mayer's reagents which indicate the presence of al~(aloids in this fraction. On

the other hand F5 was found to be positive in FeCI3 test which indicates the

presence of tannins.

The identificatio~ of compounds P1 and P2 was preliminary, using test tube

reaction. The evaluation of P1 and P2 using Anisaldehyde/H2SO4 respectively

gave the blue and the orange yellow colours while their identification using

Borntrager reaction gave the yellow and red colours respectively. These

indicate the presence of terpenoids and Quinones in P1 amd in P2 respectivelyI

(Table 3).

56

Table 3: Compounds detected in different fractions of methanol extract

QuinonesT erpenoids Alkaloids Saponins Tannins

IP1rp;

+

+

F3 + +

F4 +

Fs +

present

absent

+

5.6 Convulsant activity of pentylenetetrazole (PT;~)

Different doses of PTZ were tested for their convlJlsant inducing activity in

order to establish the dose of PTZ that will be used throughout the

anticonvulsant ,assessment experiment (Table 4). I Pentylenetetrazole

produced tonic seizures that were dose dependent. The onset of tonic

mice shortened with inc:rease in the dose ofseizu res in was an

pentylenetetrazole. The tonic proceeded by

movement, running and jerking of the mice in the (;agesj The onset of tonic

convulsions from doses of 90 mg/kg and 92.5 mgl'kg were prolonged while

those of 100 mg/kg and 95 mg/kg were short and mE~dium respectively.

intensive

seizures were

57

Table 4: Convulsant activity of pentylenetetrazole (PTZ). ip, in mice

Dose (mg/kg) No convulsed/ No used Onset of ~nic seizures (min)

rvlean I:!:: S.E.M.

8/8 9.00 1.05

8/8 8.50 0.59

95 8/8 6.25 0.67

8/8 1.75 0.62

5.7 Anticonvulsant activities of crude extracts

5.7.1 Effects of hexane, methanol and aqueDlus extracts on

pentyleneterazole- induced seizures

.The extracts obtained (HE, ME1, ME3, AQ, AQM) from plant material collected

in the summer months were tested for anticonvul~;ant activity. The hexane

extract (100 -400 mg/kg, ip) did not affect PT2~ (95 mg/kg, ip)-induced

seizures to any significant extent while the aqueous extract (100 -400 mg/kg,

ip) significantly delayed the onset of PTZ (95 mg/kg. ip)- induced seizures and

protected 12.5 -37.5 % of animals against the seizures. Both methanol

extracts (100 -400 mg/kg, ip) obtained from plants collected at the Cape Flats

Nature Reserve and Belhar significantly delayed the~ onset of PTZ (95 mg/kg,

ip)- induced seizures. In addition, 100 mg/kg (ip) of the methanol extract from

plants collected at Cape Flats Nature Reserve protel:;ted 50 % of mice against

PTZ (95 mg/kg, ip)- induced seizures while the methanol extract

(100 -200 mg/kg, ip) from plants collected at Belhar protected 37.5 % of the

animals,

58

However, the aqueous extract (100 -400 mg/kg, ip) obtairled from the residueI

after methanol extraction did not alter the seizures elicitedl by PTZ (95 mg/ kg,

ip) to any significant extent. Finally, 0.3 ml of methanol 9~.5 % and 0.2 ml of

Tween 80, both dissolved in physiological saline, did not laffect the onset norI

incidence of seizures induced by PTZ (95mg/kg, ip; -rable 15).

59

Table 5: Effects of hexane (HE), methanol (ME1. I\AE3) and aqueous (AQ,

AQM) extracts on pentylenetetrazole (PTZ)- induced !;eizures in mice.

Dose (mg/kg) No conv/

No used

Prot (%) Latency of tonic

convulsion (min)

PTZ HE AQ ME1 ME3 AQM TW MES Mean :t S.E.M.

95 8/8 0 6.25

4.88

3.50

0.37

0.85

0.33

95 100

200

8/8

8/8

0

095

95 400 - 8/8 0 3.87 0.81

13.72'"95 100 7/8 12.5 0.99

12.83""-200 6/8 25 0.6195

12.60~95 -400 5/8 37.5 0.19

17 .50~95 100 4/8 50 0.46

17 .50~6/8 25 1.0795 200

19.43'"7/8 12.5 0.6195 400

16.00~5/8 37.5 0.8395 100

17 .80~5/8 37.5 0.5295 200

20.50~95 -400 6/8 25 0.83

8/8 0 6.00 0.6895

95

100

200 8/8

8/8

0

0

7.25

6.63

0.70

0.8095 400

8/8 0

0

6.00

6.87

0.28

0.29

95 0.2 ml -

95 0.3 ml 8/8

of, : p< 0.001 compared with PTZ (95 mg/kg, ip) control, Student's t-test

AQM: Aqueous extract obtained from methanol extraj:;tion residue

ME 1: Methanol extract from plant collected at Cape Flats Nature Reserve

ME3: Methanol extract from plant collected at Belhar

TW: Tween 80

MES: Methano/99.5%

60

5.7.2 Effect of methanol extract (ME2) on PTZ. induced seizures

All the doses (100 -400 mg/kg, ip) of ME2 used did not protect any mouse

against PTZ (95 mg/kg, ip)- induced seizures. However, the onset of seizures

was significantly delayed by all the doses of methanol extract. 0.3 ml of

methanol 99.5 % dissolved in physiological saline, did not affect either the

onset or incidence of seizures induced by PTZ (95mg/kg, ip; Table 6).

Table 6: Effect of methanol extract (ME2) on PTZ- induced seizures in mice.

Dose (mg/kg) No convulsed/ Protecltion (%) Latency of tonic

No used convulsion (min)

Mean::!: S.E.M.ME2 MES

8/8 0 6.25 0.37

1 O.38~95 100 8/8 0 0.42

11.13~200 8/8 0 0.52

13.13'"400 8/8 0 0.40

a.3ml 8/8 a 6.87 0.29

40: p< 0.001 compared with PTZ induced seizures, Student's t-test

ME2: Methanol extract from plant collected during the winter month.

MES: Methano/99.5%

61

5.8 Effects of different fractions of methanol extr.act on PTZ- induced

seizures

Fractions F1, F2, F3, F4 and Fs were obtained usin~~ CC and TLC. F1 (100 -

400 mg/kg, ip) significantly delayed the latency or onset ofl PTZ (95 mg/kg, ip)-

elicited seizures. Doses of 100 mg/kg (ip), 200 mg/kg (ip) and 400 mg/kg (ip)

protected 12.5 %,50 % and 37.5 % of animals against the seizures. F2 (100-

400 mg/kg, ip) significantly delayed the onset of sei~~ures induced by PTZ (95

mg/kg, ip). Doses of 200 -400 mg/kg (ip) of F2 significantly reduced the

incidence of the seizures by protecting 87.5 ~) and 62.5 % of mice

respectively. A dose of F2 (100 mg/kg, ip) protec1:ed 5~ % of the animals

against the seizures while doses of F3 (100 -200 m~l/kg, ip) and Fs (100 -200

mg/kg, ip) did not affect PTZ (95 mg/kg, ip)- induced seizures to anysignificant extent. Finally, while a dose of F4 .--

100 mg/kg, ip) significantly

delayed the onset of seizures elicited by PTZ (95 mg/kg, ip), doses of 100

mg/kg (ip) together with 200- 400 mg/kg (ip) did not affect the incidence of the

seizure to any significant extent. 0.3 ml of methanol 99.5 % dissolved in

physiological saline, did not affect either the onset or incidence of seizures

induced by PTZ (95mg/kg, ip; Table 7).

62

Table 7: Effects of different fractions, F1, F2. F3, F4 and Fs (obtained from

methanol extract) on PTZ- induced seizures in mice

Dose (mg/kg) No conv/ protection

(%)No used

Latency of tonic

convulsion (min)

Mean :t S.E.M.PTZ F 1 F2 F3 F4 Fs MES

95 8/8 0 5.25 0.38

95 100 - 7/8 13.864012.5 1.07

95 200 4/8 21.00~50 0.7695 400 5/8 37.5 21.00~ 0.46

95 100 4/8 50 17.754 0.60

95 1/8. . 23.00.87.5 0.00-200 -

95 3/8.-400 - 23.67.62.5 0.5495

95

100 8/8 0 8.38 0.65

0.78-200 8/8 0 7.00

95 100 8/8 11.50~0 0.85

95 200 8/8 0 5.63 0.82

95 400 8/8 0 5.25 0.96

0.5695 100 - 8/8

8/8

0 5.63

5.3895

95

200 - 0 0.77

0.29-a.3ml 8/8 0 6.87

"': p< 0.001 compared with PTZ (95 mg/kg, ip) control, Student's t-test

.: p< 0.05, ..: p <0.005 compared with PTZ (95 mg/kg, ip) control,

Chi- squared test

MES: methanol 99.5 %

63

5.9 Effect of two purified products (P1 and P2) obt:ained from the

methanol extract on PTZ- induced seizures

P1 and P2 were obtained from F1 and F2 respectively using both CC and PLC.

They significantly delayed the onset of tonic convulsiion induced by PTZ (95

mg/kg, ip). At all the doses used, products P1 and P2 reduced the incidence of

PTZ (95 mg/kg, ip)-induced seizures. Product P2 at doses of 200 mg/kg (ip)

and 400 mg/kg (ip) significantly reduced the incidence of the seizures and

protected 75% (p< 0.01) and 87.5% (p<0.005) of mice respectively. Results

from P2 are the best compared with those from F'1. 0.2 ml of Tween 80,

dissolved in physiological saline, did not affect either the onset or incidence of

seizures induced by PTZ (95mg/kg, ip; Table 8).

Table 8: Effect of two purified products (P1 and P2). obtained from methanol

extract on PTZ- induced seizures in mice

No convulsed! Latency of tonicDose (mg/kg) Protection

No used (%) convulsion (min)

Mean :t S.E.M.PTZ P1 P2 TW

95 8/8 0 6.25 0.37

16.17'"95 100 6/8 25 0.35

22.00~5/8 37.5 0.4395 200

23. 75~95 400 4/8 50 0.34

20.80~5/8 37.5 1.6595 100

27 .50~2/8. 75 0.2595 200

26.00~1/8.. 0.0095 400 87.5

6.00 0.2895 0.2 ml 8/8 0

4-: p< 0.001 compared with PTZ (95 mg/kg, ip) control, Student's t-test

.: p< 0.01, ..: p< 0.005 compared with PTZ (95 mg/kg, ip) pontrol,.

Chi-squared test

64

5.10 Effect of phenobarbitone and diazepam on PTZ- induced seizures

Phenobarbitone (20 mg/kg, ip) significantly delaye(j the onset of seizures

induced by PTZ (95 mg/kg, ip) in only one animal and also significantly

reduced the incidence of the seizures. Diazepam (0.5 mgl kg, ip) effectively

protected 100 % of the animals against the seizures (Table 9).

Table 9: Effect of phenobarbitone and diazepam on I:>TZ- induced seizures in

mice

Prote(;tion

(%)

No convulsed/Dose (mg/kg) Latency of tonic

convulsion (min)No used

Mean :t S.E.M.PTZ phenobarbitone diazepam

6.18 0.2508/8

27 .oo~ 0.001/8. 87.510200.000.000/8.. 1000.5

"': p< 0.001 compared with PTZ (95 mg/kg, ip) control, Student's t-test

.: p< 0.005, ..: p< 0.001 compared with PTZ (95 mg/'kg, ip) control,

Chi-squared test

5.11 Spectra obtained from

two active com

pounds

14(}.j

40~

61l

'

~'"co(J10tit~

Figure

9: IR

spectrum

of P1

, ,

~1\!,j,JJII

,~11'\:\111,~

~1~

!1l'I

\~i~

ijlll!~

II!I~j\'r~

IJ.JJ\1~111~

ljt'rlI~

1'~1)1..1'\

1~

;I,J'I

/\

~I,lIII,

II, ~Ij'

~

MI'

I ./1\11!i (

I'I

f~I~i ! i' ".'I

I"~I

' I

Jil

l! ! 'II

b 1'1 'I

I.!U

t,..:

65

Iiti'l'r:ImijCD

I~

rfi~f~11tifi

.c

,

,I

I

II

III

"1

71

Chapter 6

DISCUSSIONS AND C:ONCLUSIONS

72

6.1 Discussions

Leonotis leonorus has been widely used to treat among other ailments,

epilepsy (Watt et al., 1962; van Wyk et al., 2000). There is little or no

scientific information on the efficacy of Leonotis leonorus in epilepsy. Claims

regarding the therapeutic success of the plant have come only from oral

communication. Thus, the need for scientific scrutirlY of the claims becomes

very necessary. Bienvenu et al. (2002) verified the anticonvulsant activity of

the plant using crude aqueous extracts. They also suggested a mechanism

for antiepileptic activity of the plant extract. No attempt has been made by any

worker to purify the compounds contained in the plant extract and to test for

antiepileptic activity in the further purified fractions. This study describes our

investigation into the antiepileptic activity of the further purified compounds in

the leaves of Leonotis leonorus. Pentylenetetrazole (PTZ) is a convulsant

drug (Rang et al., 2000) which is widely used to chemically induce

convulsions in animals. The mechanism of PTZ induced seizures in mice is

still unknown (Rang et al., 2000). According to Vida (1975), PTZ does not

block presynaptic or postsynaptic inhibition, but it activates all pathways. In.the same way according to Rang et al. (2000) the clJnvulsant and respiratory

stimulant drugs like PTZ (Ieptazol) act mainly on the brainstem and spinal

cord, producing exaggerated reflex excitability as well as an increase in

activity of the respiratory and vasomotor centres. However, De Sarro et al.

(1999) reported that PTZ produces its convulsant activity by antagonising

GABA activity in the brain. In the present study, PTZ (95 mg/kg, ip) elicited

seizures in 100 % of animals used. This compares f.3vourably with the finding

of Vida (1975) who used 85 mg/kg of PTZ to inducE~ convulsion in more than

97 % of animals.

The anticonvulsant activity assessment was preliminarily done using crude

hexane, methanol and aqueous extracts and the rE!sults obtained show that

the hexane extract did not affect PTZ induced seizures to any extent;

73

whereas both methanol and aqueous extracts were I active against the

seizures. The data obtained in this study show that the crude methanol extract

attenuated PTZ-induced seizures better than the same dose from the

aqueous extract and therefore may be more efficacious.IThis may be due to

the fact that water and methanol could be e><tracting different active

compounds according to their different polarities. For this reason, the

extraction by fractionation beginning with hexane, rnethanol and ending with

distilled water was done and followed by the anticonvulsant assessment of the

aqueous extract (AOM). This test did not show arlY anticonvulsant activity.

The latency of tonic convulsion obtained from AOrv1 inv~stigation was quite

similar to that obtained from the control experiment. It would thus appear that

all the active compounds were extracted by methanol.j The present study

shows that hexane extract has no effect on PTZ -induced seizures It is

probable that hexane did not extract any active compound(s) from the plant

material. Torssell (1983) and Lipp (1988), suggested that the composition of

some natural products in the plant extract could change according to the

season. It is not surprising, therefore, that in this s1:udy the methanol extract

(ME1) from plant material collected during the summer months demonstrated,

a better or higher anti-seizure effect against PTZ-induced seizures than the

methanol extract (ME2) from plant material collected during the winter months,

The ME1 (100 mg/kg, 200 mg/kg and 400 mg/kg) significantly prolonged the

latency of PTZ convulsions from 6.25 min to 17.50 min, 117.50 min and 19.43

min respectively. ME2 (100 mg/kg, 200 mg/kg and 400 mg/kg), on the other

hand, significantly prolonged the latency of PTZ convulsions from 6.25 min to

10.38 min, 11.13 min and 13.13 min. Reduced activity of the methanol extract

(ME2) could be due either to a decreasing concentration of active agents or, to

the increasing concentration of their antagonists in the plant. However, the

observed increase in activity when a high dose of MI=2 (400 mg/kg) was used,

supports the hypothesis that the decrease in activity of ME2 may be due to the

reduced concentration of active agents. Accordin~J to Torssell (1983), the

production of secondary metabolites is connected with several external

74

factors including season and length of daylight. The authcPr observed that the

decrease in activity of the plant material collected durirg the dark period

(winter) could be due to the degradation or translocation of active compounds

In fact, during winter, the photosynthetic activities may be reduced to the

extent that the plant could use some of the pro(jucts Isynthesised during

periods of light for its survival or protection (Tome et ai, 1995). Regarding the

origin of the plants, our study did not show any significant difference in

anticonvulsant activity between plants collected from thel Cape Flats Nature

Reserve, Bellville and Belhar. \1

Two active compounds P1 and Pz were purified as fair as possible from

methanol extract in this study. P1 which has Rf of 0.80 ir a EtOAc: Hexane

(3:7) and not soluble in water, was characterised by its dark colouration on

TLC in the visible and UV lights at 254 nm. This dark colouration is indicative

of the presence of terpenoids and flavonoids (Wa!;}ner et al., 1984). Using

anisaldehyde- sulfuric acid reagent we have stated that the compound could

also contain saponins (Wagner et al., 1984). The Carr Price test was used as

control experiment and a negative result was obtairled showing no presence.of saponins. On the other hand, the liposolubility of F)1 supports this result and

also enables us to believe that P1 may not be a flavonoid. rn fact according to

Harborne (1984) flavonoids are mainly water-soluble compounds and when

there is a partition of these, between water and an organic solvent like

petroleum ether they remain in the aqueous layer. C;ontrol experiments using

the Wilstatter reaction were carried out and the rlegative results obtained

demonstrated the absence of flavonoid material. This further supports P1 to

be a terpenoid

Strong anticonvulsant activity of terpenoids has already been shown in other

types of medicinal plants such as Delphinium denutatum~ Ginkgo biloba and

many more (Rahman et al., 1999; iHumans, inc Website, 2001). According to

McPartland et al. (2001), terpenoids could not only cross the blood brain

75

barrier (BBB) but also could act as a serotonin uptake inhibitor enhancing

norepinephrine and dopamine activity and augment gamma amino butyric

acid (GABA) concentration. It is pertinent to note that the hypothesis that the

enhancement of GABA activity in the brain, attenuates convulsions and is

widely accepted (Leidenheimer et al., 1991; Gale, K., 1992) It has furthermore

been shown that Leonotis leonorus is rich in terpenoids essentially diterpene

lactones (Rivett, 1964; Kaplan et al., 1968; Harborne et al., 1995; Hutchings

et al., 1996; van Wyk et al., 2000). A detection of P1 from a developed plate

placed in a chamber containing iodine crystals gave a brown reddish colour

while a test with concentrated H2SO4 coloured this (::ompound to green. Both

these characteristics may indicate the presence of a lactone ring in P1

(Harborne, 1984).

The IR spectrum of P1 shows strong absorption at 17'50 cm-1 and at 2950cm-1.

This shows that P1 possesses carbonyl of a lactone and saturated C-H groups

respectively (Nakanishi, 1966; Bellamy, 1975; Kemp, 1986; Pretsch et al.,

1989). In addition, the mass spectrum of P1 had major fragments at m/e 81,

m/e 109 and m/e 181 which Kaplan et al. (1968) and Habtemariam et al..(1994) have used for identifying different diterpen'e lactones isolated from

Leonotis ssp. In addition, bands at 1747 cm-1 and ~~70 cm-1 suggest a furan

moiety (Kapingu et al., 2000). The presence of furan moiety in P1 was also

confirmed by mass spectrum m/e 81 (Kaplan et al., 1968; Habtemariam et al.,

1994). An absorption at 3380 cm-1 may indicate the presence of hydroxyl

group in P1. The 1H NMR spectrum although sho'Ning peaks due to other

components did indicate the presence of p-substituted furan ring by a pair of

doublets at 7.90 and 8.00 ppm (J -2.2), (each 1 H) and a singlet at 6.24 ppm

(1 H). This doublet of doublets found at () 7.90 and 8.00 ppm indicates the AB

furan proton's while a singlet at () 6.24 ppm accounts for the third furan

proton. A 020 exchangeable singlet at () 8.54 ppm indicated the presence of

OH and multiplets in the region of() 1.10-1.85 ppm are assigned to methylene

hydrogens while sharp singlets observed at () 0.8 -0.84 ppm indicate the

76

methyl groups (Silverstein et al., 1991; Akitt, 1992). From these above results,

marrubiin (1) is strongly suspected to be the major active compound found

(P1).

0

1 marrubiin

1995) in theBiological activity has been reported by Harborne et al.

sesquiterpene lactones. Furthermore, Brown (2000) has shown that kava

lactones possess anticonvulsants properties. The anticonvulsant activity of

lactones such as massoialactone, a terpene isolated from Acollanthus

suaveotens of the family Labiatea has also been reported by Alkire (2000). In

view of these anticonvulsant activities reported for the different groups of

lactones, it is not surprising that the leaves of Leonotis leonorus, which have

been found to be rich in diterpene lactones could be used as anticonvulsant

medication in traditional medicine

Compound P2 obtained in the present study (Rf 0.66) has been classified as a

quinonoid compound. This classification is supported by its yellow colouration

on TLC and its positive test with Borntrager reaction (Santesson, 1970;

Harborne, 1984). The IR spectrum of Pz confirms the presence of carbonyl

77

group (vmax 1660- 1754 cm-1). However, 1H NMR and mars spectra obtained

from this compound were not helpful in giving informationl about the structure

of the compound since there were still impurities prE~sent that co-eluted. The

potent anticonvulsant effect of quinone has been reported by Herin et al.

(2000) who found that a quinone was able to alter the red~x modulatory site of

the NMDA receptor and was effective in limiting brairl damage in rat.

From the above results, it is tempting to suggest that Ip1 and P2 may be

exerting their anticonvulsant activity by altering both NMDA and GABA

receptor activities. This may be supported by the finding of Bienvenu et al.

(2002) who reported that the aqueous extract from Leonofis leonorus may be

exerting its anticonvulsant activity by non specifically mechanism affecting

both NMDA and GABA receptor activities. Furthermore, De Sarro et al. (1999)

has shown that PTZ may be exerting its convulsant effect by attenuating

GABAA receptor activity,

78

6.2 Conclusions

In the present study two anticonvulsant agents have been isolated in a crude

yield of 0.28 % and 0.18 % respectively. These two compounds were

classified as terpenoid lactone and as quinone com~)ound~ respectively. They

were highly lipid-soluble. This justifies the claim of tradi~ional medicine that

Leonotis leonorus was smoked for the relief of epilepsy. The spectra obtained

suggested that compound P1 may be marrubiin (1) w'hereas Pz may be

quinone. It is possible from the characterization data that Qompound P1 and Pz

exert their anticonvulsant activity by modifying both NMDA and GABA

receptor activities. Elucidation of their structures will be very important to be

able to fully investigate the toxicology, mechanism of action and so on

will help to enhance the efficacy and safety of the plants when used in

therapy. For this, the extraction must be done on large scale in order to have

sufficient quantities of active compounds for further purification and analysis.

Plant material must also be collected during thE3 summer months

season) since the plant material has been found to be the most active.

79

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